Bioinformatics and expression analysis of the Xeroderma Pigmentosum complementation group C (XPC) of Trypanosoma evansi in Trypanosoma cruzi cells / Análises de bioinformática e da expressão do gene Xeroderma Pigmentosum complementation group C (XPC) de Trypanosoma evansi em células de Trypanosoma cruzi

Abstract Nucleotide excision repair (NER) acts repairing damages in DNA, such as lesions caused by cisplatin. Xeroderma Pigmentosum complementation group C (XPC) protein is involved in recognition of global genome DNA damages during NER (GG-NER) and it has been studied in different organisms due to its importance in other cellular processes. In this work, we studied NER proteins in Trypanosoma cruzi and Trypanosoma evansi, parasites of humans and animals respectively. We performed three-dimensional models of XPC proteins from T. cruzi and T. evansi and observed few structural differences between these proteins. In our tests, insertion of XPC gene from T. evansi (TevXPC) in T. cruzi resulted in slower cell growth under normal conditions. After cisplatin treatment, T. cruzi overexpressing its own XPC gene (TcXPC) was able to recover cell division rates faster than T. cruzi expressing TevXPC gene. Based on these tests, it is suggested that TevXPC (being an exogenous protein in T. cruzi) interferes negatively in cellular processes where TcXPC (the endogenous protein) is involved. This probably occurred due interaction of TevXPC with some endogenous molecules or proteins from T.cruzi but incapacity of interaction with others. This reinforces the importance of correctly XPC functioning within the cell.

Resumo O reparo por excisão de nucleotídeos (NER) atua reparando danos no DNA, como lesões causadas por cisplatina. A proteína Xeroderma Pigmentosum complementation group C (XPC) está envolvida no reconhecimento de danos pela via de reparação global do genoma pelo NER (GG-NER) e tem sido estudada em diferentes organismos devido à sua importância em outros processos celulares. Neste trabalho, estudamos proteínas do NER em Trypanosoma cruzi e Trypanosoma evansi, parasitos de humanos e animais, respectivamente. Modelos tridimensionais das proteínas XPC de T. cruzi e T. evansi foram feitos e observou-se poucas diferenças estruturais entre estas proteínas. Durante testes, a inserção do gene XPC de T. evansi (TevXPC) em T. cruzi resultou em crescimento celular mais lento em condições normais. Após o tratamento com cisplatina, T. cruzi superexpressando seu próprio gene XPC (TcXPC) foi capaz de recuperar as taxas de divisão celular mais rapidamente do que T. cruzi expressando o gene TevXPC. Com base nesses testes, sugere-se que TevXPC (sendo uma proteína exógena em T. cruzi) interfere negativamente nos processos celulares em que TcXPC (a proteína endógena) está envolvida. Isso provavelmente ocorreu pois TevXPC é capaz de interagir com algumas moléculas ou proteínas endógenas de T.cruzi, mas é incapaz de interagir com outras. Isso reforça a importância do correto funcionamento de XPC dentro da célula.

Estudo imuno-histoquímico e classificação molecular dos adenocarcinomas de endométrio com expressão heterogênea dos genes de reparo do DNA / Immunohistochemical study and molecular classification of endometrial adenocarcinomas with heterogeneous expression of DNA repair genes

Introdução: O carcinoma endometrial (CE) foi classificado pelo sistema de Bokhman em tipos I e II com base em observações clínicas e epidemiológicas. O tipo I corresponde aos tumores de baixo grau e o tipo II aos tumores de alto grau. Adicionalmente, estudos recentes propuseram que a classificação também fosse baseada em aspectos histológicos e moleculares com base nos dados do TCGA (The Cancer Genome Atlas). Foram identificados quatro grupos moleculares distintos de CE: (1) com mutações no POLE (fenótipo "ultramutado"), (2) "alto número de cópias" (mutações em TP53), (3) !baixo número de cópias" (em que os tumores não apresentam nenhuma das alterações descritas nos outros tipos) e (4) tumores com predomínio de instabilidade de microssatélites. A imunohistoquímica (IHC) para proteínas do gene de reparo é usada para identificar a deficiência de genes de reparo do DNA (Mismatch Repair ­ MMR) associada à instabilidade de microssatélites(MSI). A coloração nuclear positiva representa a expressão retida de proteínas MMR, enquanto a perda completa representa deficiência de MMR. O padrão de expressão heterogênea (HEP), ou seja, concomitância em um mesmo espécime de áreas positivas e totalmente negativas tem sido observada em CE. No presente momento, as principais diretrizes determinam que a presença de HEP seja interpretada como expressão retida de proteínas MMR. Não há, porém, consenso quanto à classificação e interpretação de HEP, nem conhecimento do impacto da classificação de HEP como subtipo molecular diferente em relação às características clínicas e prognósticas. Objetivos: realizar a classificação molecular dos casos de CE com HEP das proteínas relacionadas aos genes de reparo do DNA e comparação do perfil molecular entre áreas positivas e negativas no estudo imunohistoquímico. Materiais e Métodos: De janeiro/2007 a dezembro/2017 foram identificados 356 casos de CE, 16 deles com HEP. A classificação molecular foi feita com base no protocolo PROMISE para CE. Cada área (expressão retida ou perdida) foi macrodissecada e o status molecular foi avaliado separadamente quanto ao status MSI (Idylla), metilação do promotor MLH1 (NGS - ponto de corte para positividade ≥ 15%), status POLE (NGS) e status p53 (IHC). Variáveis clínicas e patológicas também foram avaliadas e correlacionadas com cada caso. Resultados: A histologia endometrioide foi predominante (15 casos), bem como ausência de invasão linfovascular (11 casos), ausência de padrão MELF (10 casos), graus FIGO 1 e 2 (13 casos), invasão miometrial < 50% (13 casos) e estadiamento T1 (13 casos). Todos os pacientes estavam vivos e sem evidência de doença no último acompanhamento, exceto por um caso, cujo status de sobrevida era desconhecido. Dois casos que seriam descritos como apresentando expressão retida de proteínas relacionadas a genes de reparo do DNA por IHC apresentaram-se na análise molecular com instabilidade de microssatélites(MSI-H). Nos casos de HEP, a proteína MSH6 foi a maisfrequentemente envolvida (9 casos, 7 isolados). A proteína MLH1 apresentou-se alterada em 6 casos, sendo a única proteína associada a co-alterações (com MSH6 e PMS2). Seis casos apresentaram-se metilados por MLH1, padrão encontrado tanto em áreas com perda quanto em áreas com retenção das proteínas relacionadas a MMR por IHC e dois casos apresentaram metilação em apenas uma das áreas. Em relação ao status de POLE, 6 casos apresentaram mutação, 2 com mutações tanto em áreas com perda quanto em áreas com retenção de expressão, 3 apenas na área com perda e 1 apenas na área com retenção. Dois casos apresentam padrão aberrante de p53 (MSH6 alterados) em ambas as áreas. Conclusão: em pacientes portadoras CE e com tumores apresentando HEP a correlação entre a IHC e os achados moleculares é heterogênea e o diagnóstico entre casos com retenção ou das proteínas relacionadas a MMR não é factível apenas com realização de IHC. A análise molecular deve ser realizada em todos os casos de CE com HEP para determinar adequadamente as característicasintrínsecas de cada tumor. Devido à raridade desse achado, esta proposta é financeiramente viável e tem o potencial de mudar a prática clínica em um subconjunto de pacientes, permitindo tratamentos inovadores. HEP deve ser relatado como um padrão distinto e não considerado como uma expressão sinônimo de expressão retida de proteínas MMR em CE.

Introduction: Endometrial adenocarcinoma is classified by the Bokhman system in type I and II based on clinical and epidemiological observations, whereas the type I represents low grade tumors and type II high grade tumors. Additionally, a classification based on histological aspects and molecular profile has been proposed. The TCGA (The Cancer Genome Atlas) identified four molecular groups of endometrial adenocarcinomas: (1) mutations in POLE ("ultramutated" phenotype), (2) "high copy number" (mutations in TP53), (3) "low number of copies " (in which the tumors do not exhibit any of the changes described in the other types) and (4) tumors with predominance of microsatellite instability. In a small number of patients, heterogeneous staining is observed in the evaluation protein expression for mismatch repair genes. Objectives: to evaluate and perform the molecular classifications of cases of endometrial carcinoma with heterogeneous staining by IHC of proteins related to mismatch repair genes and comparison of the molecular profile of positive and negative areas in the IHC study. Cases and Methods: From January/2007 to December/2017 354 cases with EC were identified, 16 of those with HEP. Molecular classification was made based on the PROMISE protocol for EC. Each area (retained and lost expression) was macrodissected and molecular status was evaluated separately regarding MSI status (Idylla), MLH1 promoter methylation (NGS - cutoff for positivity ≥ 15%), POLE status (NGS) and p53 status (IHC). Clinical and pathologic variables were also evaluated and correlated with each case. Results: Endometrioid histology was predominant (15 cases), as absent lymphovascular invasion (11 cases), absence of MELF pattern (10 cases), FIGO Grade 1 and 2 (13 cases), and T1 stage (13 cases). All patients were alive and disease-free at the last follow-up. Two cases that would be described as retained by IHC presented in the molecular analysis as MSI-H. In HEP cases MSH6 was more frequent (9 cases, 7 isolated). MLH1 was altered in 6 cases, and wasthe only protein associated with co-alterations (with MSH6 and PMS2). Six cases were MLH1 methylated, found both in lost and retained areas. As POLE status, there were 6 mutated cases, 2 of those with mutations both in lost and retained areas, and 3 the lost area. Two cases had p53 aberrant pattern (MSH6 altered), that was seen both in the retained and in the lost areas. Conclusion: Correlation between IHC and molecular findings is heterogeneous, and determination between retained or lost expression of MMR proteins by IHC when HEP occurs, however feasible, does not represent the actual molecular alterations. Thus, molecular analysis should be performed every case to adequately determine the intrinsic features of each tumor. Due to the rarity of this finding, this is financially viable and has the potential to change clinical practice in a subset of patients. HEP should be reported as a distinct pattern, and not considered as a synonym expression of retained expression of MMR proteins in EC.

The molecular control of meiotic double-strand break (DSB) formation and its significance in human infertility / 亚洲男科学杂志(英文版)

Meiosis is an essential step in gametogenesis which is the key process in sexually reproducing organisms as meiotic aberrations may result in infertility. In meiosis, programmed DNA double-strand break (DSB) formation is one of the fundamental processes that are essential for maintaining homolog interactions and correcting segregation of chromosomes. Although the number and distribution of meiotic DSBs are tightly regulated, still abnormalities in DSB formation are known to cause meiotic arrest and infertility. This review is a detailed account of molecular bases of meiotic DSB formation, its evolutionary conservation, and variations in different species. We further reviewed the mutations of DSB formation genes in association with human infertility and also proposed the future directions and strategies about the study of meiotic DSB formation.

DNA repair pathways and cisplatin resistance: an intimate relationship

The main goal of chemotherapeutic drugs is to induce massive cell death in tumors. Cisplatin is an antitumor drug widely used to treat several types of cancer. Despite its remarkable efficiency, most tumors show intrinsic or acquired drug resistance. The primary biological target of cisplatin is genomic DNA, and it causes a plethora of DNA lesions that block transcription and replication. These cisplatin-induced DNA lesions strongly induce cell death if they are not properly repaired or processed. To counteract cisplatin-induced DNA damage, cells use an intricate network of mechanisms, including DNA damage repair and translesion synthesis. In this review, we describe how cisplatin-induced DNA lesions are repaired or tolerated by cells and focus on the pivotal role of DNA repair and tolerance mechanisms in tumor resistance to cisplatin. In fact, several recent clinical findings have correlated the tumor cell status of DNA repair/translesion synthesis with patient response to cisplatin treatment. Furthermore, these mechanisms provide interesting targets for pharmacological modulation that can increase the efficiency of cisplatin chemotherapy.

Mutación fundadora en síndrome de Lynch tipo II / Founder mutation in Lynch syndrome

El síndrome de Lynch es la más frecuente de las neoplasias colorrectales hereditarias. Se origina por mutaciones germinales deletéreas familia-específicas en los genes que codifican proteínas de reparación del ADN: MLH1 (homólogo humano de mutL), MSH2 y MSH6 (homólogo humano de mutS 2 y 6, respectivamente), PMS2 (homólogo humano de PMS1 2) y MUTYH (homólogo humano de la ADN-glycosilasa mutY). La mutación c.2252_2253delAA, p.Lys751Serfs*3 en el exón 19 del gen MLH1 segrega con un haplotipo descripto en la región norte de Italia y cuyo origen fue atribuido a un efecto fundador. Esta mutación co-segrega con características típicas del síndrome de Lynch, incluyendo afectación temprana y múltiples tumores primarios en el mismo individuo, una alta frecuencia de cáncer pancreático, elevada inestabilidad microsatelital y falta de expresión de PMS2. En el presente trabajo se comunica dicha mutación en una paciente argentina con adenocarcinoma endometroide de útero en cuya historia familiar existen antecedentes de cáncer de colon diagnosticado antes de los 50 años en familiares de primer grado, reuniendo los criterios de Ámsterdam I y síndrome de Lynch II. Los polimorfismos presentes en la paciente coinciden con el haplotipo descripto en una región del norte de Italia. El alto grado de patogenicidad asociada a esta mutación hace imprescindible el estudio de todos los integrantes de las familias con cáncer hereditario permitiendo el diagnóstico genético pre-sintomático, la instauración de tratamientos o conductas preventivas y su seguimiento.

Lynch syndrome is the most frequent syndrome in hereditary colorectal cancer, a family-specific deleterious mutations in genes encoding DNA reparation proteins: MLH1 (mutL homolog 1), MSH2, MSH6 (mutS homolog 2 y 6, respectively), PMS2 (PMS1 homolog 2, mismatch repair system component) y MUTYH (mutY DNA glycosylase).The c.2252_2253delAA, p.Lys751Serfs*3 mutation in MLH1 gene segregates with a haplotype reported in the northern region of Italy and whose origin was attributed to a founder effect. This mutation co-segregates with typical characteristics of Lynch syndrome, including early age at onset and multiple primary tumors in the same individual, a high frequency of pancreatic cancer, high microsatellite instability and lack of PMS2 expression. This report describes a mutation in an Argentinian patient with endometrioid adenocarcinoma of uterus. Her first-degree relatives had a history of colon cancer diagnosed before 50 years, fulfilling the Amsterdam Criteria I and Lynch syndrome II. The high pathogenicity associated to this mutation makes necessary the study of all members from families with hereditary cancer, allowing pre-symptomatic genetic diagnosis, early assessment and the instauration of preventive treatments.

Functional complementation of Leishmania (Leishmania) amazonensis AP endonuclease gene (lamap) in Escherichia coli mutant strains challenged with DNA damage agents

During its life cycle Leishmania spp. face several stress conditions that can cause DNA damages. Base Excision Repair plays an important role in DNA maintenance and it is one of the most conserved mechanisms in all living organisms. DNA repair in trypanosomatids has been reported only for Old World Leishmania species. Here the AP endonuclease from Leishmania (L.) amazonensis was cloned, expressed in Escherichia coli mutants defective on the DNA repair machinery, that were submitted to different stress conditions, showing ability to survive in comparison to the triple null mutant parental strain BW535. Phylogenetic and multiple sequence analyses also confirmed that LAMAP belongs to the AP endonuclease class of proteins.

Influence of bleaching and desensitizing gel on bond strength of orthodontic brackets

OBJECTIVE: The objective of this study was to assess, in vitro, the influence of bleaching gel and the use of desensitizing agent over bond strength of ceramic brackets bonded to bovine enamel. METHODS: One hundred bovine incisors were selected and randomly divided into five groups (n = 20): Group 1, control group (without bleaching); Group 2, bleached with 35% hydrogen peroxide; Group 3, bleached with 35% hydrogen peroxide (three applications, 15 minutes each) and desensitizing agent applied for 10 minutes; Group 4, bleached with 35% hydrogen peroxide for 40 minutes; Group 5, bleached with 35% hydrogen peroxide for 40 minutes with desensitizing agent applied for 10 minutes. Brackets were bonded 7 days after bleaching and submitted to shear bond strength test after 24 hours at a compression rate of 1 mm/minute. After fracture, the adhesive remnant index (ARI) was assessed under stereoscopic at 40 x magnification. Shear strength data (MPa) were submitted to one-way ANOVA and Tukey's test with significance level set at 5%. RESULTS: Group 5 (29.33 MPa) showed significantly higher bond strength than Group 1 (19.19 MPa), Group 2 (20.59 MPa) and Group 4 (23.25 MPa), but with no difference in comparison to Group 3. There was no significant difference among the other groups. The adhesive remnant index showed predominance of score 3, that is, all resin remained adhered to enamel for all groups. CONCLUSION: Bleaching with 35% hydrogen peroxide with calcium associated with desensitizing agent application produced higher bond strength values of brackets bonded to bovine enamel. .

OBJETIVO: o objetivo do presente estudo foi avaliar, in vitro, a influência do gel clareador e da utilização de dessensibilizante na resistência adesiva de braquetes cerâmicos colados ao esmalte bovino. MÉTODOS: cem incisivos bovinos foram aleatoriamente divididos em cinco grupos (n = 20). Grupo 1, sem clareamento (controle); Grupo 2, clareamento com peróxido de hidrogênio a 35%; Grupo 3, clareamento com peróxido de hidrogênio a 35%, sendo três aplicações de 15 minutos cada, e aplicação do dessensibilizante por 10 minutos; Grupo 4, clareamento com peróxido de hidrogênio a 35% durante 40 minutos; Grupo 5, clareamento com peróxido de hidrogênio a 35% durante 40 minutos e aplicação do dessensibilizante durante 10 minutos. Os braquetes foram colados sete dias após o clareamento e submetidos ao teste de resistência ao cisalhamento após 24 horas, a uma velocidade de compressão de 1mm/minuto. Após a fratura, avaliava-se o braquete, com lupa estereoscópica, com magnificação de 40x, e o Índice de Remanescente Adesivo (IRA). Os dados de resistência ao cisalhamento (MPa) foram submetidos à análise de variância e ao teste de Tukey, com nível de significância de 5%. RESULTADOS: os resultados mostraram que as amostras do Grupo 5 apresentaram resistência de união significativamente superior à dos grupos 1, 2 e 4, mas sem diferença do Grupo 3. Não houve diferença significativa entre a resistência de união dos demais grupos. O Índice de Remanescente Adesivo mostrou predominância do escore 3, ou seja, toda resina permaneceu aderida ao esmalte, para todos os grupos. CONCLUSÃO: pôde-se concluir que o clareamento com peróxido de hidrogênio a 35%, com cálcio associado à aplicação do dessensibilizante, produziu maior resistência dos braquetes ao esmalte bovino. .

Caracterization of the SOS response in Leptospira interrogans sorovar Copenhageni / Caracterização da resposta SOS em Leptospira interrogans sorovar Copenhageni

Leptospira is a basal genus in an ancient group of bacteria, the spirochetes. The pathogenic species are responsible for leptospirosis, a disease with worldwide distribution and of public health importance in developed tropical countries. L. interrogans serovar Copenhageni is the agent for the majority of human leptospirosis in Brazil. In this work, we used a great variety of experimental approaches to characterize the SOS system in this serovar, to identify its impact in general DNA damage response, as well as to assess the DNA repair toolbox owned by pathogenic and saprophytic leptospires. We identified an additional repressor LexA, acquired by lateral gene transfer, exclusively in serovar Copenhageni. We also observed that UV-C irradiation led to massive death of cells and blockage of cell division in the survivors. Both repressors were active and we identified the sequences responsible for binding to promoters. However, the LexA1 SOS box was redefined after a de novo motif search on LexA1 ChIP-seq enriched sequences. This regulator was able to bind to at least 25 loci in the genome. DNA damage also caused a massive rearrangement of metabolism: increase in expression was observed in transposon and prophage genes, in addition to DNA repair pathways and mutagenesis inducers; on the other hand, motility, general metabolism and almost all virulence genes were repressed. Two induced prophages provided several proteins with useful functions. We also assessed the DNA repair-related genes presented by the three species of Leptospira: the saprophytic L. biflexa, the facultative pathogen L. interrogans and the obligatory pathogen L. borgpetersenii. There are more diversity and redundancy of repair genes in L. interrogans in comparison with the other species. Lateral gene transfer seems to be an important supplier of DNA repair functions. In addition, leptospires share characteristics of both Gram-positives and Gram-negatives bacteria. Representative genes from several different pathways were induced during infection of susceptible mice kidneys, suggesting DNA repair genes are active while causing disease. All these data suggest mobile genetic elements are the major forces in leptospiral evolution. Moreover, during DNA damage response, several SOS-dependent and independent mechanisms are employed to decrease cell growth and virulence in favor of controlled induction of mechanisms involved in genetic variability

Leptospira é um gênero basal em um grupo já considerado um dos mais ancestrais, as espiroquetas. As espécies patogênicas são responsáveis pela leptospirose, uma doença presente em todo o mundo e de principal importância em países tropicais em desenvolvimento. L. interrogans sorovar Copenhageni é o agente da maior parte dos casos no Brasil. Nesse trabalho, utilizamos diversas abordagens experimentais para caracterizar o sistema SOS nesse sorovar, identificar seu impacto na resposta geral a danos no DNA, assim como avaliar as funções de reparo de DNA disponíveis em leptospiras patogênicas e saprofíticas. Identificamos um repressor LexA adicional, adquirido por transferência horizontal e exclusivo do sorovar Copenhageni. Observamos também que irradiação por UV-C causou significativa morte celular e bloqueio da divisão celular dos sobreviventes. Ambos os repressores são ativos e identificamos as sequências que utilizam para se ligar aos promotores dos genes regulados. Entretanto, o SOS box de LexA1 foi redefinido após uma busca de novo por motivos enriquecidos nas sequências recuperadas por ChIP-seq. Esse regulador ligou-se ao menos a 25 locais do genoma. A maioria desses alvos teve aumento de expressão após UV-C. Danos no DNA também causaram um importante rearranjo metabólico: houve aumento de expressão em transposons e profagos, além de indutores de mutagênese e vias de reparo; por outro lado, mobilidade, crescimento celular e quase todos os fatores de virulência foram reprimidos. Dois profagos induzidos durante essa resposta, possivelmente proporcionam algumas proteínas de funções importantes. Nós também avaliamos a presença de genes envolvidos no reparo de DNA em três espécies de leptospira: L. biflexa, L. interrogans e L. borgpetersenii. L. interrogans é a espécie com maior diversidade e redundância de genes de reparo. Além disso, transferência horizontal parece ser um importante fornecedor de funções de reparo nesse gênero. Leptospiras também apresentam genes característicos tanto de bactérias Gram-positivas quanto Gram-negativas. Genes representando diferentes vias de reparo foram induzidos durante infecção em modelo animal, sugerindo que essas vias estão ativas no curso da doença. Todos esses dados, em conjunto, sugerem que elementos genéticos móveis são de extrema importância na evolução do gênero e das vias de reparo. Assim, durante a resposta a danos no DNA, diversos mecanismos dependentes e independentes de SOS são empregados para frear o crescimento celular e virulência em favor da indução controlada de mecanismos para aumentar variabilidade genética

Deoxyribonucleic acid repair gene X-ray repair cross-complementing group 1 polymorphisms and non-carcinogenic disease risk in different populations: A meta-analysis.

PURPOSE: This study aims to assess a meta‑analysis of the association of X‑ray repair cross‑complementing group 1 (XRCC1) polymorphisms with the risk of various non‑carcinogenic diseases in different population. MATERIALS AND METHODS: This meta‑analysis was performed by critically reviewing reveals 38 studies involving 10043 cases and 11037 controls. Among all the eligible studies, 14 focused on Arg194Trp polymorphism, 33 described the Arg399Gln and three articles investigated on Arg280His. Populations were divided into three different ethnic subgroups include Caucasians, Asians and other (Turkish and Iranian). RESULTS: Pooled results showed no correlation between Arg194Trp and non‑carcinogenic disease. There was only weak relation in the recessive (odds ratio [OR] =1.11, 95% confidence interval [CI]: 0.86‑1.44) model in Asian population and dominant (OR = 1.04, 95% CI: 0.66‑1.63) model of other populations. In Arg399Gln polymorphism, there was no relation with diseases of interest generally. In the pooled analysis, there were weak relation in the dominant (OR = 1.08, 95% CI: 0.86‑1.35) model of Asian population and quite well‑correlation with recessive (OR = 1.49, 95% CI: 1.19‑1.88), dominant (OR = 1.23, 95% CI: 0.94‑1.62), and additive (OR = 1.23, 95% CI: 0.94‑1.62) models of other subgroup. For Arg280His, there was a weak relation only in the dominant model (OR = 1.06, 95% CI: 0.74‑1.51). CONCLUSION: The present meta‑analysis correspondingly shows that Arg399Gln variant to be associated with increased non‑carcinogenic diseases risk through dominant and recessive modes among Iranian and Turkish population. It also suggests a trend of dominant and recessive effect of Arg280His variant in all population and its possible protective effect on non‑carcinogenic diseases.

Xeroderma pigmentoso: comunicación de un caso / Xeroderma pigmentosum: report of a case

El xeroderma pigmentoso (XP) es una rara enfermedad, autosómica recesiva, causada por un defecto en la reparación del ADN. Se manifiesta en la niñez temprana. Los pacientes conXP tienen una extrema fotosensibilidad, lo que conduce a quemaduras solares, cambios pigmentarios y una elevada incidencia de tumores malignos de piel. Una minoría de pacientes presenta anomalías neurológicas progresivas. El diagnóstico es clínico y puede ser confirmado con estudios genéticos. El tratamiento consiste en una rigurosa fotoprotección y la extirpación precoz de lesiones precancerosas. Presentamos el caso de un niño de 2 años de edad con compromiso cutáneo y ocular, sinalteraciones neurológicas...

DNA Repair Genes XRCC1, XRCC3, XPD, and OGG1 Polymorphisms among the Central Region Population of Saudi Arabia

DNA repair is one of the central defense mechanisms against mutagenic exposures. Inherited SNPs of DNA repair genes may contribute to variations in DNA repair capacity and susceptibility to cancer. Due to the presence of these variants, inter-individual and ethnic differences in DNA repair capacity have been established in various populations. Saudi Arabia harbors enormous genetic and cultural diversity. In the present study we aimed to determine the genotype and allele frequencies of XRCC1 Arg399Gln (rs25487), XRCC3 Thr241Met (rs861539), XPD Lys751Gln (rs13181), and OGG1 Ser326Cys (rs1052133) gene polymorphisms in 386 healthy individuals residing in the central region of Saudi Arabia and compare them with HapMap and other populations. The genotype and allele frequencies of the four DNA repair gene loci in central Saudi population showed a distinctive pattern. Furthermore, comparison of polymorphisms in these genes with other populations also showed a unique pattern for the central Saudi population. To the best of our knowledge, this is the first report that deals with these DNA repair gene polymorphisms among the central Saudi population.

Implication of DNA repair genes in prostate tumourigenesis in Indian males.

Background & objectives: Prostate cancer (CaP) is the fifth most common cancer among Indian men. Tumour protein p53 (TP53) gene increases the fidelity of DNA replication and homologous recombination by transcriptional transactivation of mismatch repair (MMR) genes. DNA repair thus has a potential role in molecular carcinogenesis of CaP. The aim of the present study was to identify mutations, and polymorphisms in TP53 gene and MMR protein expression in CaP in Indian male population. Methods: TP53 codon 72 polymorphism was analysed in 105 CaP, 120 benign prostatic hyperplasia (BPH) cases and 106 normal controls. Mutational analysis of TP53 was done in DNA extracted from formalin fixed paraffin embedded tissue of 80 CaP and 24 BPH cases. Expression of MMR proteins viz. hMLH1, hMSH2, hPMS1 and hPMS2 was studied in 80 CaP, 15 prostatic intraepithelial neoplasia (PIN) and 15 BPH cases. Results: A somatic C/A variation at the intronic boundary of exon 7 in TP53 gene was observed in one each biopsy samples from CaP and BPH. A significant association of codon 72 TP53 Pro/Pro genotype was observed with the risk of CaP (OR, 2.59, P=0.02) and BPH (OR, 6.27, P<0.001). Immunohistochemical analysis of MMR proteins showed maximum loss of hPMS1 expression in cases of CaP and PIN while no loss in expression of MMR proteins was observed in BPH cases. The study also identified a significant loss of hPMS2 protein in poorly differentiated tumours (Gleason score >7) than in well differentiated tumours (Gleason score 3-6) (P<0.05). Interpretation & conclusions: The results of the present study demonstrate that TP53 codon 72 polymorphism plays significant role in the pathogenesis and susceptibility to CaP and BPH. Also, an aberrant MMR protein expression could be involved in progression of prostate cancer through PIN, early CaP to aggressive CaP. The loss of hPMS2 protein expression may serve as a marker for progression of CaP.

Síndrome de Lynch: selección de pacientes para el estudio genético mediante análisis de inestabilidad microsatelital e inmunohistoquímica / Lynch syndrome: selection of families by microsatellite instability and immunohistochemistry

Background: Selection of patients with Lynch Syndrome (LS) for a genetic study involves the application of clinical criteria. To increase the rate of identification of mutations, the use of molecular studies as Microsatellite Instability (MSI) and Im-munohistochemistry (IHC) in the tumor has been proposed. Aim: To demonstrate the usefulness of MSI and IHC in the detection of mutations in patients with LS. Material and Methods: From our Familial Colorectal Cancer Registry, families suspected of LS were selected according to Amsterdam or Bethesda clinical criteria. Screening of germline mutations of MLH1, MSH2 and MSH6 genes was performed. In addition, analysis of MSI and IHC were performed in colorectal tumors. Results: A total of 35 families were studied (19 met Amsterdam and 16 met Bethesda criteria). Twenty one families harbored a germline alteration in MLH1, MSH2 or MSH6 (18 Amsterdam and 3 Bethesda). In these families, eighteen different alterations were found, 15 of which were mutations and 3 corresponded to variants of uncertain pathogenicity. On the other hand, 80% of the tumors showed positive microsatellite instability (27 MSI-high and 1 MSI-low), and immunohistochemical testing showed that 77% of tumors had the loss of a protein. Correlation between results of tumor molecular studies and the finding of germline nucleotide change showed that IHC and MSI predicted mutations in 81 and 100% of patients, respectively. Conclusions: MSI and IHC can efficiently select patients with a high probability of carrying a mutation in DNA repair genes.

Polymorphisms in base-excision & nucleotide-excision repair genes & prostate cancer risk in north Indian population.

Background & objectives: Genetic variation in the DNA repair genes might be associated with altered DNA repair capacities (DRC). Reduced DRC due to inherited polymorphisms may increase the susceptibility to cancers. Base excision and nucleotide excision are the two major repair pathways. We investigated the association between two base excision repair (BER) genes (APE1 exon 5, OGG1 exon 7) and two nucleotide excision repair (NER) genes (XPC PAT, XPC exon 15) with risk of prostate cancer (PCa). Methods: The study was designed with 192 histopathologically confirmed PCa patients and 224 age matched healthy controls of similar ethnicity. Genotypes were determined by amplification refractory mutation specific (ARMS) and PCR-restriction fragment length polymorphism (RFLP) methods. Results: Overall, a significant association in NER gene, XPC PAT Ins/Ins (I/I) genotype with PCa risk was observed (Adjusted OR- 2.55, 95%CI-1.22-5.33, P=0.012). XPC exon 15 variant CC genotypes presented statistically significant risk of PCa (Adjusted OR- 2.15, 95% CI-1.09-4.23, P=0.026). However, no association was observed for polymorphism with BER genes. Diplotype analysis of XPC PAT and exon 15 revealed that the frequency of the D-C and I-A diplotype was statistically significant in PCa. The variant genotypes of NER genes were also associated with high Gleason grade. Interpretation & conclusions: The results indicated that there was a significant modifying effect on the association between genotype XPC PAT and exon 15 polymorphism and PCa risk which was further confirmed by diplotype analysis of XPC PAT and exon 15 in north Indian population.

Genetic variation in nucleotide excision repair pathway genes influence prostate and bladder cancer susceptibility in North Indian population.

BACKGROUND: Inherited polymorphisms of XPD and XPC genes may contribute to subtle variations in NER DNA repair capacity and genetic susceptibility to development of urological cancer such as prostate and bladder cancer. MATERIALS AND METHODS: We genotyped four Single Nucleotide Polymorphs (SNPs) of the DNA repair gene XPD and XPC in 195 prostate cancer (PCa) and 212 bladder cancer (BC) patients and 250 healthy controls from the same area. XPD Exon 10 (G>A) by amplification refractory mutation system and Exon 23 (A>C), XPC Intron 9 (Ins/Del) and Exon 15 (A>C) were genotyped by PCR-RFLP. RESULTS: Variant genotype of XPC demonstrated association with PCa as well as in BC (P, 0.013; P, 0.003). Combined genotype (GA+AA) revealed association with PCa and in BC (P, 0.012, P, 0.002). Variant allele also demonstrated risk in both the cancer. Diplotype of XPD and XPC was associated with a significant increase in PCa and BC risk. Variant (+/+) genotype of XPC intron 9 shown increased risk with PCa and in BC (P, 0.012; P, 0.032). CC genotype of XPC exon 15 revealed increase risk (P, 0.047) with PCa not in BC. In clinopathological grade variant allele of XPC intron 9 and 15 demonstrated risk with high grade of tumor and bone metastasis of PCa. In BC variant allele of XPD exon 10 and 15 also shown association with tumor grade. XPC intron 9 influences the risk of BC in former tobacco users in BC. CONCLUSIONS: Our result support that SNPs in XPD and XPC gene may reduce NER repair capacity and play a major role for PCa and BC in North India.

Efeito da p53 sobre a expressão e atividade da enzima de reparo de DNA Timina-DNA Glicosilase / Effect of p53 on the expression and activity of DNA repair enzyme thymine-DNA glycosylase

O câncer de esôfago é uma malignidade altamente freqüente e letal. Uma característica específica das áreas de alta incidência de câncer de esôfago é a grande proporção de duplas mutações no gene TP53, sendo, ao menos uma delas, uma transição G para A em sítios CpG. Essas transições resultam de malpareamentos G.T causados pela desaminação espontânea da 5-metilcitosina em ilhotas CpG. A enzima de reparo de DNA Timina-DNA Glicosilase (TDG) é responsável pelo primeiro passo na remoção da timina de malpareamentos G.T em CpG. A alta proporção de mutações em sítios CpG em câncer de esôfago das áreas de alta incidência sugere que a via de reparo de DNA iniciada pela TDG pode estar prejudicada. A presença de duplas mutações, sendo ao menos uma delas em CpG, levantou a hipótese de que a primeira mutação no TP53 reduz a atividade da via de reparo iniciada pela TDG, que acarretaria a segunda mutação em sítios CpG. Dessa forma, o objetivo desse trabalho foi analisar o efeito da p53 sobre a expressão e atividade da TDG. Os resultados obtidos mostram que a expressão de TDG é regulada transcricionalmente pela p53 numa gama de linhagens celulares e é induzida pelo dano ao DNA, de forma p53-dependente. Além disto, os resultados apontam um possível papel da proteína p53 ativa na migração nuclear e atividade da TDG. Estes resultados ainda nos levam à conclusão de que o silenciamento de TDG aumenta a sensibilidade à morte celular induzida por MMS quando a p53 é encontrada na forma selvagem, mas não quando esta proteína é mutada, e de que o status mutacional de TP53 parece afetar a expressão de TDG em CEE primários. Juntos esses resultados sugerem que a p53 regula o reparo de DNA mediado pela TDG e que a inativação de p53 em células tumorais pode contribuir para a aquisição de um mutator phenotype

Esophageal squamous cell carcinoma (ESCC) is a highly frequent and fatal malignancy in the world. A peculiar characteristic of the high incidence areas of esophageal cancer is the large proportion of double mutations in TP53 gene, being, at least one of them, a G to A transition at CpG sites. These transitions result from G.T mismatches caused by the spontaneous deamination of 5-methylcytosine at CpG sites. The DNA repair enzyme Thymine-DNA Glycosylase (TDG) is responsible for the first step in the removal of the thymidine from the G.T mismatches at CpG sites. The high proportion of mutations at CpG sites in esophageal tumors in the high incidence areas suggests that the DNA repair pathway initiated by TDG might be impaired. The large number of double mutations, with one being at a CpG site, raised the possibility that the first mutation in TP53 reduces the activity of the TDG base excision repair pathway, increasing the chance of a second mutation event at a CpG site. In this way, the aim of this work was to analyze the effect of p53 on the expression and activity of TDG. The results achieved show that TDG expression is regulated by p53 in a variety of cells lines at the trancriptional level and induced by DNA–damage in a p53-dependent manner. Furthermore, these results point out a possible role of active p53 in the nuclear migration and activity of TDG. The results further support the notion that TDG silencing increases the sensitivity to cell death induced by Methylmethane sulphonate when p53 is found in a wild-type, but not in a mutant form, and that TP53 mutation seems to affect TDG expression in primary ESCC. Together, these results suggest that p53 regulates TDG-mediated repair and that p53 inactivation in cancer cells may contribute to a mutator phenotype through loss of TDG function

R659X mutation in the MLH1 gene in hereditary non-polyposis colorectal cancer (HNPCC) in an Indian extended family.

Background & objective: Hereditary non-polyposis colorectal cancer (HNPCC or Lynch syndrome), is a genetically heterogeneous disorder that is believed to account for 2–10 per cent of all the colorectal cancer cases. The disease follows autosomal dominant inheritance pattern with high penetrance (85%) and younger age of onset when compared to patients with sporadic tumours. HNPCC is associated with germ-line mutations in the DNA mismatch repair (MMR) genes namely MLH1, MSH2, MSH6, and PMS2. The present study was aimed at analyzing mismatch repair gene(s) in an extended Indian family satisfying the Amsterdam criteria, and extending the analysis to general population to estimate frequency of the mutations/polymorphisms observed. Methods: A total 12 members of the HNPCC family were studied for genetic investigation. Ethnically matched 250 normal individuals were also included as controls to study the observed mutations/ polymorphisms at population level. Results: The analysis resulted in identification of a 1975C>T mutation in exon 17, resulting in substitution of arginine residue with stop codon at codon 659. 655A>G substitution was also observed, resulting in replacement of isoleucine with valine at codon 219. Similar analysis on 250 ethnically matched control subjects revealed complete absence of R659X mutation, while I219V variant was found in 9.8 per cent of the controls. Interpretation & conclusion: R659X mutation correlates with disease phenotype, and 655A>G locus is highly polymorphic. Our study suggested that R659X substitution was prime cause for the disease phenotype in this family. I219V substitution is a polymorphism having no association with the disease onset or segregation. The family members harbouring this mutation were advised to be under regular medical surveillance.

Participação de enzimas de reparo por excisão de bases na restauração de lesões induzidas pela associação da radiação ultravioleta A e cloreto estanoso em Escherichia coli / Participation of repair enzyme for base excision in lesions restoration induced by the combination of A ultraviolet radiation and stannous chloride in Escherichia coli

O cloreto estanoso (SnCI2) e a radiação ultravioleta A (UVA) são agentes que lesam diversas estruturas celulares, inclusive o DNA, principalmente pela geração de espécies reativas de oxigênio. O objetivo deste trabalho foi estudar a mutagênese e o reparo das lesões produzidas pela combinação do UVA, na condição de pré-iluminação, com o SnCI2. Avaliou-se a ação de enzimas do reparo por excisão de bases (BER), em Escherichia coli (E. coli), por eletroforese em gel alcalino de agarose e sobrevivência bacteriana. Também se estudou a indução do sistema SoxRS pelo cromoteste, e a mutagênese pelo teste de Ames. De acordo com os resultados: i)o UVA induziu quebras no DNA das cepas testadas e os mutantes fpg-nfo e fpg apresentaram maior retardo no reparo das lesões; ii) o SnCI2 induziu mais quebras que o UVA e os mutantes nfo e fpg mostraram maior dificuldade em reparar as lesões; iii) o UVA+SnCI2 provocou mais quebras que o SnCI2 e os mutantes nfo e fpg também apresentaram maior lentidão no reparo das lesões; iv) o UVA não inativou as cepas testadas; v) as cepas nfo e fpg foram as mais sensíveis ao SnCI2; vi) o UVA+SnCI2 provocou maior letalidade em todas as cepas testadas, em relação ao SnCI2, e os mutantes nfo e fpg também foram os mais sensíveis ao tratamento com ambos os agentes; vii) a transformação dos mutantes nfo com o plasmídio pBW21 (nfo+) e dos mutantes fpg com o plasmídio pFPG (fpg+) aumentou a sobrevivência das cepas aos tratamentos com SnCI2 e UVA+SnCI2; viii) o SnCI2 induziu o sistema SoxRS; ix) o SnCI2, UVA e UVA+SnCI2 não induziram mutagênese; x) o reparo das lesões parece ser preferencialmente realizado pelas proteínas Fpg e Nfo.

Stannous chloride (SnCI2) and ultraviolet radiation A (UVA) are able to induce lesions in different cellular structures, including DNA, manly through ROS generation. The aim of this work was to study the mutagenesis and repair of lesions induced by the association of UVA (pre treatment) with SnCI2. It was evaluated the action of base excision repair (BER) enzymes in Escherichia coli (E. coli) by alkaline gel electrophoresis and bacterial survival. It was also evaluated the SoxRS system induction by chromotest and mutagenesis through the Ames test. According to the results: i) UVA induced DNA strand breaks in all strains and fpg-nfo and fpg mutants showed greater delay in the repair of lesions; ii) SnCI2 induced more breaks than UVA and nfo and fpg mutants showed more difficult to repair the damage; iii) UVA + SnCI2 caused more breaks than the SnCI2 and nfo and fpg mutants also showed a slowest repair of injuries; iv) UVA did not inactivate any bacterial strains tested; v) nfo and fpg strains were more sensitive to SnCI2; vi) UVA + SnCI2 caused higher mortality in all strains tested, when compared to SnCI2, and, again, nfo and fpg mutants were the most sensitives to the treatment with both agents; vii) the transformation of nfo mutant with the plasmid pBW21 (nfo+) and fpg mutants with plasmid pFPG (fpg+) increased the survival of the strains to SnCI2 and UVA + SnCI2 treatments; viii) SnCI2 was able to induce SoxRS system; ix) SnCI2, UVA + SnCI2 and UVA did not induce mutagenesis; x) damage repair seems to be preferentially performed by Fpg and Nfo proteins.

Multiple Roles of BRIT1/MCPH1 in DNA Damage Response, DNA Repair, and Cancer Suppression

Mammalian cells are frequently at risk of DNA damage from both endogenous and exogenous sources. Accordingly, cells have evolved the DNA damage response (DDR) pathways to monitor and assure the integrity of their genome. In cells, the intact and effective DDR is essential for the maintenance of genomic stability and it acts as a critical barrier to suppress the development of cancer in humans. Two central kinases for the DDR pathway are ATM and ATR, which can phosphorylate and activate many downstream proteins for cell cycle arrest, DNA repair, or apoptosis if the damages are irreparable. In the last several years, we and others have made significant progress to this field by identifying BRIT1 (also known as MCPH1) as a novel key regulator in the DDR pathway. BRIT1 protein contains 3 breast cancer carboxyl terminal (BRCT) domains which are conserved in BRCA1, MDC1, 53BP1, and other important molecules involved in DNA damage signaling, DNA repair, and tumor suppression. Our in vitro studies revealed BRIT1 to be a chromatin-binding protein required for recruitment of many important DDR proteins (ATM, MDC1, NBS1, RAD51, BRCA2) to the DNA damage sites. We recently also generated the BRIT1 knockout mice and demonstrated its essential roles in homologous recombination DNA repair and in maintaining genomic stability in vivo. In humans, BRIT1 is located on chromosome 8p23.1, where loss of hetero-zigosity is very common in many types of cancer. In this review, we will summarize the novel roles of BRIT1 in DDR, describe the relationship of BRIT1 deficiency with cancer development, and also discuss the use of synthetic lethality approach to target cancers with HR defects due to BRIT1 deficiency.

Multiple Roles of BRIT1/MCPH1 in DNA Damage Response, DNA Repair, and Cancer Suppression

Mammalian cells are frequently at risk of DNA damage from both endogenous and exogenous sources. Accordingly, cells have evolved the DNA damage response (DDR) pathways to monitor and assure the integrity of their genome. In cells, the intact and effective DDR is essential for the maintenance of genomic stability and it acts as a critical barrier to suppress the development of cancer in humans. Two central kinases for the DDR pathway are ATM and ATR, which can phosphorylate and activate many downstream proteins for cell cycle arrest, DNA repair, or apoptosis if the damages are irreparable. In the last several years, we and others have made significant progress to this field by identifying BRIT1 (also known as MCPH1) as a novel key regulator in the DDR pathway. BRIT1 protein contains 3 breast cancer carboxyl terminal (BRCT) domains which are conserved in BRCA1, MDC1, 53BP1, and other important molecules involved in DNA damage signaling, DNA repair, and tumor suppression. Our in vitro studies revealed BRIT1 to be a chromatin-binding protein required for recruitment of many important DDR proteins (ATM, MDC1, NBS1, RAD51, BRCA2) to the DNA damage sites. We recently also generated the BRIT1 knockout mice and demonstrated its essential roles in homologous recombination DNA repair and in maintaining genomic stability in vivo. In humans, BRIT1 is located on chromosome 8p23.1, where loss of hetero-zigosity is very common in many types of cancer. In this review, we will summarize the novel roles of BRIT1 in DDR, describe the relationship of BRIT1 deficiency with cancer development, and also discuss the use of synthetic lethality approach to target cancers with HR defects due to BRIT1 deficiency.